Hemostatic composition and hemostatic method using hemostatic composition

ABSTRACT

[Problem] To provide a composition for hemostasis that can be applied uniformly to a bleeding site and exerts a high hemostatic effect. [Solution] Provided is a composition to be applied to the subject as a spray, wherein the composition is characterized in that the spray is for hemostasis, and the composition includes a self-assembling peptide, the self-assembling peptide gelling due to self-assembly when the composition is applied to the bleeding site of the subject, and the self-assembling peptide being included in the composition in a concentration having an improved hemostatic capacity in comparison to direct application.

TECHNICAL FIELD

The present invention relates to a spray for hemostasis comprising aself-assembling peptide.

BACKGROUND ART

Biogels such as fibrin glue are utilized in perioperative orpostoperative hemostats, surgical base materials such as wound healingsheets, drug delivery systems (DDSs), etc.

However, most of these biogels employ organism-derived materials andtherefore have the risk of infection by microbes such as viruses,immunogenicity, propagation of diseases, etc. when used. For example,fibrin glue, albeit highly useful as a perioperative hemostat, hasbecome a significant social problem due to frequent accidents causingthe infection of patients by hepatitis virus contaminants in the fibringlue when actually used in operation, because its raw material isderived from human blood. Accordingly, there is a demand for thedevelopment of a safe hemostat without the risk of microbialcontamination by the chemical synthesis of its raw material.

For example, use of a composition comprising a purified amphipathicself-assembling peptide in the promotion of wound healing, etc. has beenproposed so far (Patent Literature 1).

CITATION LIST Patent Literature

-   Patent Literature 1: National Publication of International Patent    Application No. 2008-505919

SUMMARY OF INVENTION Technical Problem

The self-assembling peptide composition as described in PatentLiterature 1 is a very useful composition because this composition canpromote tissue regeneration by application to a wound site (or anoperative site) and has no risk of microbial contamination.

The present inventors have tackled application research on theself-assembling peptide as described above and consequently found thatin the case of using the self-assembling peptide as described above forhemostasis (e.g., for a hemostat for use in surgical operation), thereis a technical dilemma, i.e., a higher concentration of the peptide in acomposition to be applied to a bleeding site improves a hemostaticeffect, but elevates the viscosity of the composition to be applied sothat the applied composition is difficult to spread at the bleeding siteand thus has the difficulty in exerting a uniform hemostatic effect atthe application site (in other words, a lower concentration of thepeptide in the composition to be applied to a bleeding site facilitatesspreading the applied composition at the bleeding site, but does notproduce a high hemostatic effect).

The present inventors have conducted diligent studies on a method forovercoming the technical dilemma described above and consequentlycompleted the present invention by finding that, surprisingly, by usinga composition comprising a self-assembling peptide as a “spray”, thecomposition comprising a self-assembling peptide can be uniformlyapplied to a bleeding site and produces a high hemostatic effect ascompared with that when applied by a conventional method.

Solution to Problem

Specifically, in one embodiment, the present invention relates to acomposition to be applied as a spray to a subject, the spray being usedfor hemostasis, and the composition comprising a self-assemblingpeptide, wherein the self-assembling peptide self-assembles and therebygels when the composition is applied to a bleeding site of the subject,and the self-assembling peptide is contained in the composition at aconcentration at which the composition has an improved hemostaticability as compared with that when directly applied.

In one embodiment of the present invention, the concentration of theself-assembling peptide in the composition is at least 1.5% by weight orhigher.

In one embodiment of the present invention, the self-assembling peptideis

(a) a peptide comprising an amino acid sequence consisting of 4 to 34amino acid residues, wherein polar amino acid residues and nonpolaramino acid residues are alternately arranged, or

(b) a peptide comprising an amino acid sequence consisting of 13 aminoacid residues, wherein nonpolar amino acids and polar amino acids arearranged alternately and at symmetric positions in directions toward theN terminus and the C terminus centered on a nonpolar amino acid residueat position 7.

In one embodiment of the present invention, the polar amino acidresidues are amino acid residues selected from the group consisting ofan aspartic acid residue, a glutamic acid residue, an arginine residue,a lysine residue, a histidine residue, a tyrosine residue, a serineresidue, a threonine residue, an asparagine residue, a glutamineresidue, and a cysteine residue.

In one embodiment of the present invention, the nonpolar amino acidresidues are amino acid residues selected from the group consisting ofan alanine residue, a valine residue, a leucine residue, an isoleucineresidue, a methionine residue, a phenylalanine residue, a tryptophanresidue, a proline residue, and a glycine residue.

In one embodiment of the present invention, the polar amino acidresidues are amino acid residues selected from the group consisting ofan aspartic acid residue, a glutamic acid residue, an arginine residue,a lysine residue, a histidine residue, a tyrosine residue, a serineresidue, a threonine residue, an asparagine residue, a glutamineresidue, and a cysteine residue, and the nonpolar amino acid residuesare amino acid residues selected from the group consisting of an alanineresidue, a valine residue, a leucine residue, an isoleucine residue, amethionine residue, a phenylalanine residue, a tryptophan residue, aproline residue, and a glycine residue.

In one embodiment of the present invention, the self-assembling peptideis a peptide comprising RADARADARADARADA (SEQ ID NO: 1), a peptidecomprising IEIKIEIKIEIKI (SEQ ID NO: 2), or a peptide comprisingRLDLRLALRLDLR (SEQ ID NO: 3).

In one embodiment of the present invention, the self-assembling peptideis a peptide consisting of RADARADARADARADA (SEQ ID NO: 1), a peptideconsisting of IEIKIEIKIEIKI (SEQ ID NO: 2), or a peptide consisting ofRLDLRLALRLDLR (SEQ ID NO: 3).

In one embodiment of the present invention, the bleeding site of thesubject is a non-surgically formed wound site or a surgically formedincision site or excision site.

In an alternative embodiment, the present invention relates to use ofany of the compositions described above for the production of a sprayfor hemostasis.

In an alternative embodiment, the present invention relates to a spraycomprising any of the compositions described above.

In an alternative embodiment, the present invention relates to a methodfor hemostasis in a subject, comprising the step of applying acomposition comprising a self-assembling peptide to a bleeding site ofthe subject by spraying, wherein the self-assembling peptideself-assembles and thereby gels when the composition is applied to thebleeding site of the subject, and the self-assembling peptide iscontained in the composition at a concentration at which the compositionhas an improved hemostatic ability as compared with that when directlyapplied.

In one embodiment of the present invention, the subject is a nonhumananimal.

Any combination of one or more of the features of the present inventiondescribed above is also included in the scope of the present invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a syringe packed with a peptide solution, used in Examplesof the present invention.

FIG. 2 shows photographs taken after application of Peptide No. 1 (1.0%or 1.5%) to a bleeding site of the porcine liver by usual application orspray application.

FIG. 3 shows photographs taken after application of Peptide No. 1 (2.0%or 2.5%) to a bleeding site of the porcine liver by usual application orspray application.

FIG. 4 shows photographs taken after application of Peptide No. 2 (1.0%or 1.5%) to a bleeding site of the porcine liver by usual application orspray application.

FIG. 5 shows photographs taken after application of Peptide No. 3 (1.5%)to a bleeding site of the porcine liver by usual application or sprayapplication.

FIG. 6 shows results of measuring a hemostasis completion time and theamount of a composition applied until the completion of hemostasis, whenPeptide No. 1 (1.0%, 1.5%, 2.0%, or 2.5%) was applied to a bleeding siteof the porcine liver by usual application or spray application.

FIG. 7 shows results of measuring the amount of a composition applieduntil the completion of hemostasis when Peptide No. 2 (1.0% or 1.5%) wasapplied to a bleeding site of the porcine liver by usual application orspray application.

FIG. 8 shows results of measuring a hemostasis completion time whenPeptide No. 2 (1.0% or 1.5%) was applied to a bleeding site of theporcine liver by usual application or spray application.

FIG. 9 shows results of measuring a hemostasis completion time and theamount of a composition applied until the completion of hemostasis, whenPeptide No. 3 (1.5%) was applied to a bleeding site of the porcine liverby usual application or spray application.

DESCRIPTION OF EMBODIMENTS

The present invention relates to a composition to be applied as a sprayto a subject. In the present invention, the “spray” means an agent thatis applied in a spray form to a recipient subject. In the presentinvention, a method, an instrument or an apparatus for applying thecomposition in a spray form is not limited and can be appropriatelyselected by those skilled in the art. The composition can be applied ina spray form, for example, by spraying a liquid composition in a statesuch as a mist or a foam using compressed air or high-pressure gas. Morespecifically, the composition can be applied in a spray form, forexample, through the use of a can spray based on internal high-pressuregas, an electric spray based on an electric pump or the like, or an airspray (e.g., a spray gun or an air brush) based on external airpressure.

The present invention also relates to an agent for hemostasis. In thepresent invention, the agent “for hemostasis” broadly means an agentthat is used for arresting bleeding from a living body and includes notonly an agent that completely arrests bleeding but an agent thatalleviates bleeding.

The bleeding site which is subject to the hemostasis according to thepresent invention may be any site. The present invention can be used in,for example, the hemostasis of a non-surgically formed wound site or asurgically formed incision site or excision site. Examples of thenon-surgically formed wound site can include wound sites of body surfacetissues (e.g., the skin) or body tissues (e.g., organs, muscles, andbones) caused by accidents, etc. Examples of the surgically formedincision site or excision site can include sites of body surface tissuesor body tissues incised or excised with a surgical knife or the like insurgical procedures, and sites of body surface tissues or body tissuesexfoliated, anastomosed, sutured, etc. with a surgical instrument. Thepresent invention is applied to a subject by spraying and thereforepermits easier application operation than that of conventional productsin such a way that the composition is uniformly applicable to a widerange by single operation. Thus, the present invention can also besuitably used for a bleeding site resulting from, for example,endoscopic operation which requires careful operation.

The recipient subject of the present invention can be a human ornonhuman subject. The nonhuman subject can be, for example, a nonhumananimal and can be, for example, a nonhuman mammal, bird, a reptile, anamphibian, or fish. Examples of the nonhuman mammal include rodents(e.g., mice and rats), dogs, cats, horses, pigs, cattle, sheep, goat,and primates

The composition of the present invention comprises a self-assemblingpeptide. In the present specification, the phrase “peptide“self-assembles” in a solution” means that the molecules of the peptideassemble spontaneously via any interaction (e.g., electrostaticinteraction, a hydrogen bond, a van der Waals' force, or hydrophobicinteraction) in the solution, and must not be interpreted in a limitedmeaning. In the present invention, the self-assembling peptide means apeptide having a property of forming a self-assembly form in which manypeptide molecules are regularly arranged according to their amino acidsequences. Owing to this property, when the composition comprising theself-assembling peptide is applied to a bleeding site of the subject,the self-assembling peptide self-assembles and thereby forms a gel atthe application site.

The self-assembling peptide used in the present invention may have givenviscosity of its aqueous solution (i.e., an aqueous peptide solutionbefore self-assembly of the self-assembling peptide) itself beforeapplication to the subject. However, in the present specification, suchan aqueous peptide solution before application to the subject is alsoreferred to as a “peptide solution (or aqueous peptide solution)” evenwhen having given viscosity, for the sake of convenience of explanation.Also, even when the aqueous peptide solution before application to thesubject has given viscosity, further elevation in the viscosity of thecomposition by the self-assembly of the self-assembling peptide afterapplication of the aqueous solution to the subject is also referred toas “gelling (or gel formation)”.

The self-assembling peptide used in the present invention can be, forexample, a peptide comprising an amino acid sequence consisting of 4 to34 amino acid residues, wherein polar amino acid residues and nonpolaramino acid residues are alternately arranged, or a peptide comprising anamino acid sequence consisting of 13 amino acid residues, whereinnonpolar amino acids and polar amino acids are arranged alternately andat symmetric positions in directions toward the N terminus and the Cterminus centered on a nonpolar amino acid residue at position 7.

By having the peptide structure as described above, the self-assemblingpeptide used in the present invention forms a β sheet structure in anaqueous solution such that only the polar amino acid residues can bearranged on one surface of the β sheet structure while only the nonpolaramino acid residues can be arranged on the other surface. Thus, such βsheet structures are capable of forming a two-layer structure byassembling so as to conceal their hydrophobic surfaces (surfaces onwhich only the nonpolar amino acid residues are arranged). Furthermore,as the self-assembly of the molecules proceeds, this layer structure ofthe β sheets is capable of elongating to form a three-dimensionalconformational structure (e.g., a hydrogel). The “self-assemblingpeptide” used in the present invention is also referred to as a“self-organizing peptide” in some cases.

The self-assembling peptide used in the present invention may comprisean “amino acid sequence in which polar amino acid residues and nonpolaramino acid residues are alternately arranged”. In this case, the aminoacid sequence can be an amino acid sequence consisting of 4 to 34 aminoacid residues, more preferably an amino acid sequence consisting of 8 to30 amino acid residues, further preferably an amino acid sequenceconsisting of 12 to 26 amino acid residues, most preferably an aminoacid sequence consisting of 13 to 20 amino acid residues.

In the present invention, the “amino acid” is used in the broadest senseand includes not only a protein-constituting amino acid but anon-protein-constituting amino acid such as an amino acid variant andderivative. Those skilled in the art should understand in considerationof this broad definition that examples of the amino acid according tothe present invention include: protein-constituting L-amino acids;D-amino acids; chemically modified amino acids such as amino acidvariants and derivatives; non-protein-constituting amino acids such asnorleucine, β-alanine, and ornithine; and chemically synthesizedcompounds having properties known in the art as features of the aminoacid. Examples of the non-protein-constituting amino acid includeα-methylamino acids (α-methylalanine, etc.), D-amino acids,histidine-like amino acids (2-amino-histidine, β-hydroxy-histidine,homohistidine, α-fluoromethyl-histidine and α-methyl-histidine, etc.),amino acids having extra methylene in a side chain (“homo” amino acids)and amino acids with a carboxylic acid functional group amino acid, in aside chain, substituted by a sulfonic acid group (cysteic acid, etc.).In a preferred aspect of the present invention, the amino acid used inthe present invention can be a protein-constituting amino acid.

In the present invention, the polar amino acid residues are notparticularly limited as long as the polar amino acid residues are aminoacid residues whose side chain can have polarity. The polar amino acidresidues include, for example, acidic amino acid residues and basicamino acid residues. In the present specification, the acidic amino acidresidues include, for example, an aspartic acid (Asp: D) residue andglutamic acid (Glu: E). The basic amino acids include, for example,arginine (Arg: R), lysine (Lys: K), and histidine (His: H).

In the present specification, for example, the term “aspartic acid (Asp:D)” means that a three-letter code “Asp” and a one-letter code “D” maybe used as the abbreviations of the aspartic acid.

In the present specification, among neutral amino acid residues, aminoacid residues containing a hydroxy group, an acid amide group, a thiolgroup, or the like are included in the polar amino acid residues becauseof having polarity. In the present specification, for example, tyrosine(Tyr: Y), serine (Ser: S), threonine (Thr: T), asparagine (Asn: N),glutamine (Gln: Q), and cysteine (Cys: C) are included in the polaramino acid residues.

In the present specification, the nonpolar amino acid residues are notparticularly limited as long as the nonpolar amino acid residues areamino acids whose side chain has no polarity. The nonpolar amino acidresidues include, for example, alanine (Ala: A), valine (Val: V),leucine (Leu: L), isoleucine (Ile: I), methionine (Met: M),phenylalanine (Phe: F), tryptophan (Trp: W), glycine (Gly: G), andproline (Pro: P).

When the self-assembling peptide used in the present invention comprisesan “amino acid sequence in which polar amino acid residues and nonpolaramino acid residues are alternately arranged”, this peptide can have arepeat sequence of “RADA” (2 to 8 repeats, preferably 3 to 6 repeats) ora repeat sequence of “IEIK” (1 to 4 repeats, preferably 2 to 3 repeats)and can be more preferably a peptide comprising RADARADARADARADA (SEQ IDNO: 1) or a peptide comprising IEIKIEIKIEIKI (SEQ ID NO: 2). Furtherpreferably, the self-assembling peptide used in the present inventioncan be a peptide consisting of RADARADARADARADA (SEQ ID NO: 1) or apeptide consisting of IEIKIEIKIEIKI (SEQ ID NO: 2).

When the self-assembling peptide used in the present invention is a“peptide comprising an amino acid sequence consisting of 13 amino acidresidues, wherein nonpolar amino acids and polar amino acids arearranged alternately and at symmetric positions in directions toward theN terminus and the C terminus centered on a nonpolar amino acid residueat position 7”, preferably, the “nonpolar amino acid residue at position7” of this peptide can be alanine (Ala: A). More preferably, the peptidecan be a peptide comprising RLDLRLALRLDLR (SEQ ID NO: 3), furtherpreferably, a peptide consisting of RLDLRLALRLDLR (SEQ ID NO: 3).

Other examples of the self-assembling peptide which can be used in thepresent invention can include peptides disclosed in WO2006/014570.

The self-assembling peptide used in the present invention may bemodified (or labeled) as long as the peptide does not lose the mainproperties intended by the present invention. Such a modified (orlabeled) peptide is also included in the “self-assembling peptide”according to the present invention. A method for modifying (or labeling)the self-assembling peptide used in the present invention can bearbitrarily selected by those skilled in the art. For example, theattachment of a functional group or the like, the attachment of achemical substance, or the attachment of an additional protein orpeptide can be performed. Examples of the attachment of a functionalgroup or the like can include acylation, acetylation, alkylation,amidation, biotinylation, formylation, carboxylation, glutamylation,glycosylation (attachment of a sugar chain), glycylation, hydroxylation,isoprenylation, lipoylation, the attachment of a nucleotide or itsderivative, polyethylene glycolation (PEGylation), and the attachment ofa lipid chain. Examples of the attachment of a chemical substance caninclude the attachment of an appropriate labeling agent, for example,radioisotopes (e.g., ¹²⁵I, ¹³¹I, ³H, and ¹⁴C), enzymes (e.g.,β-galactosidase, β-glycosidase, alkaline phosphatase, peroxidase, andmalate dehydrogenase), fluorescent materials (e.g., fluorescamine andfluorescein isothiocyanate), luminescent materials (e.g., luminol,luminol derivatives, luciferin, and lucigenin), and affinity tags (e.g.,biotin). Examples of the attachment of an additional protein or peptidecan include ISGylation, SUMOylation, and ubiquitination.

The concentration of the self-assembling peptide in the composition ofthe present invention can be at least approximately 0.5% by weight, atleast approximately 1% by weight, at least approximately 1.5% by weight,at least approximately 2% by weight, at least approximately 2.5% byweight, at least approximately 3% by weight, at least approximately 3.5%by weight, at least approximately 4% by weight, at least approximately4.5% by weight, at least approximately 5% by weight, at leastapproximately 5.5% by weight, at least approximately 6% by weight, atleast approximately 6.5% by weight, at least approximately 7% by weight,at least approximately 7.5% by weight, or at least approximately 8% byweight. Preferably, the concentration of the self-assembling peptide inthe composition of the present invention can be at least approximately1.5% by weight.

The composition of the present invention may further contain one or morebiologically active substances (e.g., substances useful in hemostasisand substances useful in the promotion of wound healing) withoutsubstantially influencing the ability of the self-assembling peptide toself-assemble in the composition. Examples of such substances includesynthetic or natural organic molecules, nucleic acid molecules, variouscytokines, and various chemokines. A growth factor may also be containedalone or in combination with a different biologically active substancein the composition of the present invention. Examples of the growthfactor include, but are not limited to, epidermal growth factors, nervegrowth factors, transforming growth factors alpha and beta,platelet-derived growth factors, insulin-like growth factors, vascularendothelial growth factors, hematopoietic cell growth factors, heparinbinding growth factors, acidic fibroblast growth factors, basicfibroblast growth factors, hepatocyte growth factors, brain-derivedneurotrophic factors, keratinocyte growth factors, bone morphogenicproteins, and cartilage-derived growth factors.

The terms used in the present specification are given for illustratingparticular embodiments and are not intended to limit the invention.

The term “comprising” used in the present specification means thatdescribed items (members, steps, factors, numbers, etc.) are present andthe presence of the other items (members, steps, factors, numbers, etc.)is not excluded therefrom, unless the context evidently requiresdifferent interpretation.

All the terms (including technical terms and scientific terms) usedherein have the same meanings as those understood in a broad sense bythose skilled in the art to which the present invention belongs, unlessotherwise defined. The terms used herein should be interpreted as havingmeanings consistent with those in the present specification and relatedtechnical fields, unless otherwise defined, and should not beinterpreted in an idealized or excessively formal sense.

Terms such as “first” or “second” may be used for expressing variousfactors. However, it is understood that these factors should not belimited by these terms. These terms are used merely for differentiatingone factor from the other factors. For example, the first factor may bedescribed as the second factor, and vice versa, without departing fromthe scope of the present invention.

Hereinafter, the present invention will be described more specificallywith reference to Examples. However, the present invention can beembodied in various forms and must not be interpreted as being limitedto Examples described herein.

EXAMPLES <Object of Test>

Oozing bleeding was created by abrading the porcine liver using a metalfile. A self-organizing peptide solution was applied to the bleeding byusual application or spray application and evaluated for its hemostaticeffect and applied amount.

<Guideline of Test Practice>

This test was conducted as a non-GLP test.

<Animal Protection>

This test was conducted with the approval of the Institutional AnimalCare and Use Committee according to “rules and regulations for thecontact of animal experiments” provided by NAS Laboratory Co., Ltd. asto the items of animal experiments to be practiced from a scientificstandpoint and under ethical consideration, on the basis of “Act onWelfare and Management of Animals (Act No. 105 of 1973)”, “StandardsRelating to the Care and Management of Laboratory Animals and Relief ofPain (Notice. No. 88 of the Ministry of the Environment, 2006)”, “BasicPolicies for Conduct of Animal Experiments” of the Ministry ofAgriculture, Forestry and Fisheries and the Ministry of Health, Labourand Welfare, and “Guidelines for Proper Conduct of Animal Experiments(Science Council of Japan, Jun. 1, 2006)”.

<Test Animal>

Animal species: pig

Breed: LWD

Sex: femaleThe number of animals introduced: 2Age in days at the time of introduction: anyBody weight at the time of introduction: 50 kg Institute receiving theanimals: Narita experimental laboratory of NAS Laboratory Co., Ltd.Distributor of the animals: Ishige TikusanAcclimatization period: approximately 7 days after introductionQuarantine: the absence of abnormalities was confirmed by quarantineunder clinical observation at the time of introduction and during theacclimatization period.Individual discrimination: ear tag

<Rearing Conditions> (1) Rearing Environment

The test animals were each individually raised in a stainless cage (W:690×D: 960×H: 740 mm) of a rearing room with a temperature and ahumidity set. The temperature and the humidity were controlled using anair conditioner set to 25±3° C. and 60±10%, respectively. Illuminationwas set to 12-hour light and 12-hour dark periods (light period: 7:00 to19:00). The cleaning of the cage and a feeder and the cleaning anddisinfection of the rearing room were carried out every day.

(2) Type of Feed, Amount of Feed and Feeding Method

The feed used was “Breed 74” (JA Higashinihon Kumiai Shiryo Co., Ltd.)and was given to the animals twice a day (AM and PM) in an amount of 250g per meal. However, on the day of the experiment, the animals were notfed in order to avoid misswallowing due to vomiting when anesthetized,etc.

(3) Drinking of Water

For feeding of water, the animals were allowed to freely drink wellwater using an automatic feed-water apparatus. Substance concentrationsand bacterial counts in the drinking water were analyzed once a year,and the analysis values were confirmed to conform to the water qualitycriteria of the Water Supply Act.

<Individual Discrimination and Constitution of Test Group>

The individual discrimination of the animals was performed using ear tagnumbers. The entrances of the rearing rooms were marked with testnumbers and test periods, and the cages indicated test numbers, testgroups, and animal numbers.

<Pretreatment and Anesthetic Administration Method>

Atropine sulfate was intramuscularly administered at 0.05 mg/kg (dosingvolume: 0.1 mL/kg) to each animal. Approximately 30 minutes later, 20mg/kg (dosing volume: 0.4 mL/kg) of ketamine hydrochloride and 4 mg/kg(dosing volume: 0.2 mL/kg) of xylazine hydrochloride were mixed andintramuscularly administered thereto. Each anesthetic was introduced bythe intramuscular administration to the ear root using a disposablesyringe (Terumo Corp.) attached with an 18 G injection needle (TerumoCorp.). Then, a tracheal tube (TOP intratracheal tube, TOP Corp.) wasintubated, and the anesthesia was maintained with isoflurane using aninhalation anesthesia apparatus SUBARU-KJ02 (Mikasa Kantou Syoukai Co.,Ltd.) and a mechanical ventilator (COMPOS β-EV, Metran Co., Ltd.). Theperioperative isoflurane concentration was set to 1 to 3%, and theamount of oxygen was set to 2 L/min. The intrapulmonary pressure of themechanical ventilator was set to 15 cmH2O, and the respiratory rate wasset to 15 times/min. Each animal was placed supine on an operating tableand covered with a sterilized drape.

<Perioperative Monitoring>

An electrocardiogram, a pulse oximeter, and a thermometer were set up,and perioperative monitoring was carried out. Blood pressure wasbloodlessly measured by setting up a cuff for blood pressuremeasurement. The blood pressure was maintained at an average bloodpressure of 60 mmHg or higher.

<Test Substance>

The peptide solutions (test substances) used in this experiment andtheir storage elastic moduli are shown in the table below.

TABLE 1 Before application Storage elastic modulus G′ (Pa) (mean: N = 3)SD Peptide No. 1 1.0% (pH no treatment) 30 3.9 Peptide No. 1 1.5% (pH notreatment) 75 9.5 Peptide No. 1 2.0% (pH no treatment) 141 8.1 PeptideNo. 1 2.5% (pH no treatment) 503 22.4 Peptide No. 2 1.0% (pH 3.0) 349.045.5 Peptide No. 2 1.5% (pH 3.0) 2,309 50.5 Peptide No. 3 1.0% (pH 7.5)75 5.4 Peptide No. 3 1.5% (pH 7.5) 239 19.9 Peptide No. 3 2.0% (pH 7.5)439 12.1 (Peptide concentration: % by weight)

(SEQ ID NO: 1) Peptide No. 1: RADARADARADARADA (SEQ ID NO: 2)Peptide No. 2: IEIKIEIKIEIKI (SEQ ID NO: 3) Peptide No. 3: RLDLRLALRLDLR

For All the peptides described above, the bulk powder was synthesized byCPC Scientific, Inc. All the peptides were N-terminally acetylated andC-terminally amidated. Purified water of Japanese Pharmacopoeia was usedas a solvent for the peptides.

Frequency sweep tests were conducted from 1 rad/sec to 10 rad/sec at ashear stress of 1 Pa, and the storage elastic modulus at 10 rad/sec wasselected as data. As for a viscosity before gelling, tests wereconducted for a soaking time of 2 min by loading each peptide solutionbetween 20 mm plates with a gap distance of 500 um.

<Testing Method>

1. Each completely anesthetized pig was laparotomized by abdominalmidline incision using a surgical knife and an electric surgical knifeto expose the liver.2. Oozing bleeding was created by abrading the liver using a metal file.3. Each test substance was applied to the bleeding inclined plane byusual application (*1) or spray application (*2).4. The time from after the end of application to the end of assessmentof the completion of hemostasis was measured using a stopwatch (*3). Theamount of each test substance used until the end of the test wasrecorded.*1: A syringe was packed with the peptide solution (test substance), andthe peptide solution was pushed out of a nozzle at the tip of thesyringe and thereby applied to the subject (see the syringe of FIG. 1).*2: A syringe (equivalent to that of FIG. 1) packed with the peptidesolution (test substance) was loaded in a spray gun, and compressed airwas blown to a nozzle at the tip of the syringe using a compressor sothat the peptide solution pushed out of the nozzle was sprayed to thesubject. The spray pressure was generally 0.5 to 1.5 [kgf/cm2], thoughdiffering depending on the viscosity of each test substance, the statusof bleeding, and the distance to the application site.*3: If bleeding was not weakened after initial application (if it wasjudged that this condition would not lead to hemostasis), additionalapplication was appropriately performed. The time of completion ofinitial application was defined as the start of measurement, and thetime of completion of hemostasis assessment was defined as the end ofmeasurement.

RESULTS

In the tests described above, the way in which each peptide solution wasapplied to the bleeding site of the liver is shown in FIGS. 2 to 5. Asseen in the drawings, the peptide solution was nonuniformly spread overthe bleeding site by the usual administration, whereas the peptidesolution was uniformly spread over the bleeding site by the sprayapplication.

As shown in FIG. 6, the peptide solution containing Peptide No. 1 at theconcentration of 1.0% had tendencies of a shorter hemostasis completiontime and a smaller amount of the solution applied until hemostasis, inthe spray application group than in the usual application group, thoughthere was no significant difference. On the other hand, theconcentrations of 1.5% or higher evidently resulted in a shorterhemostasis completion time and a smaller amount of the solution applieduntil hemostasis, in the spray application group than in the usualapplication group.

As shown in FIGS. 7 and 8, the peptide solution containing Peptide No. 2at the concentration of 1.0% also exhibited no significant difference inresults between the spray application group and the usual applicationgroup, as in the results about the peptide solution containing PeptideNo. 1. By contrast, the concentration of 1.5% evidently resulted in ashorter hemostasis completion time and a smaller amount of the solutionapplied until hemostasis, in the spray application group than in theusual application group.

As shown in FIG. 9, the peptide solution containing Peptide No. 3 alsosucceeded in hemostasis in the spray application group, but failed inhemostasis by the usual application.

These results demonstrated that, surprisingly, a solution containing theself-assembling peptide, when used as a spray, exerts an improvedhemostatic ability as compared with that when usually administered.

1. A composition to be applied as a spray to a subject, the spray beingused for hemostasis, and the composition comprising a self-assemblingpeptide, wherein the self-assembling peptide self-assembles and therebygels when the composition is applied to a bleeding site of the subject,and the self-assembling peptide is contained in the composition at aconcentration at which the composition has an improved hemostaticability as compared with that when directly applied.
 2. The compositionaccording to claim 1, wherein the concentration of the self-assemblingpeptide in the composition is at least 1.5% by weight or higher.
 3. Thecomposition according to claim 1, wherein the self-assembling peptide is(a) a peptide comprising an amino acid sequence consisting of 4 to 34amino acid residues, wherein polar amino acid residues and nonpolaramino acid residues are alternately arranged, or (b) a peptidecomprising an amino acid sequence consisting of 13 amino acid residues,wherein nonpolar amino acids and polar amino acids are arrangedalternately and at symmetric positions in directions toward the Nterminus and the C terminus centered on a nonpolar amino acid residue atposition
 7. 4. The composition according to claim 3, wherein the polaramino acid residues are amino acid residues selected from the groupconsisting of an aspartic acid residue, a glutamic acid residue, anarginine residue, a lysine residue, a histidine residue, a tyrosineresidue, a serine residue, a threonine residue, an asparagine residue, aglutamine residue, and a cysteine residue.
 5. The composition accordingto claim 3, wherein the nonpolar amino acid residues are amino acidresidues selected from the group consisting of an alanine residue, avaline residue, a leucine residue, an isoleucine residue, a methionineresidue, a phenylalanine residue, a tryptophan residue, a prolineresidue, and a glycine residue.
 6. The composition according to claim 3,wherein the polar amino acid residues are amino acid residues selectedfrom the group consisting of an aspartic acid residue, a glutamic acidresidue, an arginine residue, a lysine residue, a histidine residue, atyrosine residue, a serine residue, a threonine residue, an asparagineresidue, a glutamine residue, and a cysteine residue, and the nonpolaramino acid residues are amino acid residues selected from the groupconsisting of an alanine residue, a valine residue, a leucine residue,an isoleucine residue, a methionine residue, a phenylalanine residue, atryptophan residue, a proline residue, and a glycine residue.
 7. Thecomposition according to claim 6, wherein the self-assembling peptide isa peptide comprising RADARADARADARADA (SEQ ID NO: 1), a peptidecomprising IEIKIEIKIEIKI (SEQ ID NO: 2), or a peptide comprisingRLDLRLALRLDLR (SEQ ID NO: 3).
 8. The composition according to claim 7,wherein the self-assembling peptide is a peptide consisting ofRADARADARADARADA (SEQ ID NO: 1), a peptide consisting of IEIKIEIKIEIKI(SEQ ID NO: 2), or a peptide consisting of RLDLRLALRLDLR (SEQ ID NO: 3).9. The composition according to claim 1, wherein the bleeding site ofthe subject is a non-surgically formed wound site or a surgically formedincision site or excision site.
 10. (canceled)
 11. A spray comprisingthe composition of claim
 1. 12. A method for hemostasis in a subject,comprising the step of applying a composition comprising aself-assembling peptide to a bleeding site of the subject by spraying,wherein the self-assembling peptide self-assembles and thereby gels whenthe composition is applied to the bleeding site of the subject, and theself-assembling peptide is contained in the composition at aconcentration at which the composition has an improved hemostaticability as compared with that when directly applied.
 13. The methodaccording to claim 12, wherein the concentration of the self-assemblingpeptide in the composition is at least 1.5% by weight or higher.
 14. Themethod according to claim 12, wherein the bleeding site of the subjectis a non-surgically formed wound site or a surgically formed incisionsite or excision site.
 15. The method according to claim 12, wherein thesubject is a nonhuman animal.